1. Field of the Invention
This invention relates generally to methods and devices used for determination of analytes in bodily fluids, and in particular, pertains to a sensor strip for determination of organic and ionic constituents in tears. Analyzing the constituents of tears lends itself to frequent home monitoring because of the non-invasiveness of the method.
2. Description of the Related Art
Bodily fluids such as blood are often analyzed for trace constituents as being symptomatic of the physiological and medical health of patients. Obtaining blood for the tests, however, is an invasive measure requiring arterial or venous puncture, which is preferably avoided. The means of analysis of these bodily fluid vary, and include various analytical devices and electrometric measurement means. The constituents found in tears are representative of those found in the blood supply to the brain, because the palpebral conjunctiva is supplied by the ophthalmic artery, a branch of the internal carotid artery, a major supplier of the brain. Although tears are an alternative body fluid which can be analyzed, it is generally difficult or impossible to obtain a large enough tear sample to allow measurement or detection of constituents. To obtain such a volume of tears for research or analysis, investigators have generally been required to use artificial stimulation of tear production, for example, with tear-inducing chemicals, fans, and the like. The concentration of some solutes in tears is flow-dependent and therefore depends on the method of collection of the tears.
Schirmer tear strips (SNO*STRIPS.TM.) can be ordered from Smith and Nephew Pharmaceuticals (Romford, England). The Schirmer tear strips are sterile tear flow test strips (60 mm.times.5 mm) cut off from Whatman (no.41) filter paper absorbent material. The strips are folded 5 mm from the rounded end, and the folded end is inserted into the inferior conjunctival sac at the junction of the middle and temporal thirds of the eyelids. The subject closes the eyes lightly for five minutes and the strips are removed and the extent of wetting is measured from the fold.
The method and device of the invention allows determination of such organic tear constituents such as glucose, urea, and ketone bodies, as well as the determination of ionic tear constituents such as potassium, magnesium and calcium. The need for determination of these constituents, as well as previous monitoring techniques, are discussed below.
Glucose. The glucose concentration of tears remains invariant until the blood sugar exceeds the threshold level. When the level of plasma glucose rises to 200 mg/dl or 10 mM it may be correlated with the hyperglycemic elevated levels, and tear glucose can be used as an index for blood glucose concentration. See, Van Harringen et al., Albret von Graefes Arch. Klin. Ophthalmol. 202:1, 1977). The method of the invention allows home monitoring of glucose in the tear fluid as a substitute for fractional urine glucose determination as a method of guiding insulin therapy in diabetes mellitus.
Urea. The concentration of urea in tears (TUN) has been shown to be in close agreement with the simultaneous blood values of urea nitrogen (BUN), implying an unrestricted passage of the urea molecule through the blood-tear barrier in the lacrimal gland. Therefore, TUN can be monitored non-invasively as a substitute for BUN. The method of the invention allows assessment, in a clinical or other setting, of the adequacy of dialysis (both hemodialysis and peritoneal dialysis) in end-stage renal disease, as well as monitoring the development of uremia as the patient progresses from renal insufficiency to renal failure.
Ketone bodies. Free fatty acids from adipose tissue stores are the source of ketone bodies. Ketosis or ketogenesis is the process of ketone body formation. Ketogenesis is stimulated by glucagon excess and insulin lack as in diabetic keto acidosis (DKA). Ketone bodies appear in blood and urine in DKA and starvation. In DKA treatment they disappear from the urine early. The end point for correction of DKA is the disappearance of ketone bodies from the blood. Tears, which are more representative of the environment of the brain than is systemic blood is the ideal non-invasive fluid to monitor for ketone body appearance and disappearance. Currently, ketone bodies are determined semi-quantitatively in the blood or urine by reagent test strips (for example, KETOSTIX.TM., made by Ames, Division of Miles Laboratories). Ketone bodies are also determined by ACETEST.TM. tablets (manufactured by Ames, Division of Miles Laboratories) which are made of glycine, Na-nitroprusside, Na.sub.2, PO.sub.4, and lactose, are dissolved in the fluid to be tested. A positive result with these tablets yields a lavender purple color when the concentration of ketone bodies is greater than or equal to 5-10 mg%.
Potassium ion. Potassium disorders range from hyperkalemia to hypokalemia, and are second only to hydrogen ion disorders as causes of mortality and morbidity. Hyperkalemia is the most dangerous and lethal electrolyte disorder and constitutes a common cause of sudden death on account of fatal cardiac arrhythmias, but may be reversed if appropriate measures are taken in time. Hyperkalemia, in which the serum K.sup.+ concentration is greater than 5.5 mM/l can be caused by excessive potassium intake, inadequate K.sup.+ excretion or a shift of K.sup.+ from the tissues to extracellular fluid (ECF). There are a number of commonly used drugs which contribute to the latter two causes. The heart is very sensitive to changes in serum K.sup.+, and is effectively a bio-sensor for extracellular K.sup.+ levels. With hyperkalemia, cardiac excitability increases, which is reflected in EKG changes which have some correlation with the K.sup.+ level.
Hypokalemia, in which the serum K.sup.+ level is less than 3.5 mM/l, is often associated with K.sup.+ depletion. Hypokalemia and K.sup.+ depletion can be caused by inadequate K.sup.+ intake, excess renal K.sup.+ excretion, excessive gastrointestinal (GI) K.sup.+ loss by vomiting or diarrhea, and a shift of K.sup.+ into cells and tissues. The clinical manifestations of hypokalemia are cardiac (predisposition to digitalis intoxication and irregular heartbeats), hemodynamic (decrease in blood pressure), neuromuscular weakness and paralysis of GI and striated/skeletal muscle, renal (inability to concentrate urine progressing to renal diabetes insipidus), and endocrine (decrease in renin and aldosterone, and decrease in insulin secretion with carbohydrate intolerance of diabetes).
Though the instantaneous determination (STAT) of serum K.sup.+ has saved lives, many crises are missed due to the lack of a non-invasive, monitoring system which is easily used and may be used at home. Tears contain about 15-30 milliequivalents (mEq) of K.sup.+ per liter, which is about 3-6 times the level of serum K.sup.+ , with an average level in serum being about 4.5 mEq/liter. This indicates that there is active secretion of K.sup.+ in tears (Thaysen et al., Am. J. Physiol. 178:160, 1954; Miller, Am. J. Opthalmol. 47:773, 1970).
Calcium and Magnesium. Blood plasma is quite different from the interstitial fluid that bathes the cells directly. Plasma is a colloidal solution while interstitial fluid is a simple crystalloid solution. Blood plasma is continuously stirred and agitated and mixed with an almost equal volume of mostly charged red blood cells. Electrometric determination of ionic activity in plasma, which is a currently widely accepted method of determining Ca.sup.2+, is problematic, first, due to plasma proteins, because of a phenomenon known as residual liquid junction at the tip of the reference electrode when it measures a constituent of plasma against simple crystalloid standard solutions, and second, due to the sedimentation of charged red blood cells in whole blood due to the influence of gravity.
Because tears do not have plasma proteins, they are closer to interstitial fluids than plasma. Interstitial fluid calcium is critical for a variety of functions especially neuromuscular excitability and irritability. Low free Ca.sup.2+ and Mg.sup.2+ cause convulsions and tetany. In addition, low Ca.sup.2+ levels are commonly seen in conditions of low plasma proteins in newborns and alkalosis. Magnesium deficiency may be associated with normo-calcemic or hypocalcemic states, as well as with hyperirritability, tetany, convulsions and EKG changes, and is often associated with K.sup.+ deficiency.
Magnesium and calcium in blood plasma occur in three forms: (1) free ions; (2) diffusible complexes; and (3) protein-bound. One-half of the calcium is protein-bound, while 1/3 of the total magnesium is protein-bound, with most of the remaining calcium and magnesium being ionic Ca.sup.2+ and Mg.sup.+2, and the complexes being only a few percent.
Thus, an electrometric determination of ionic Ca.sup.2+ and Mg.sup.+2 in tears is more reliable than in plasma, and correlates better with clinical neuromuscular irritability, in view of the effect of proteins on the residual liquid junction and the low (about 0.3) activity coefficient of the solution (activity coefficient being defined as the activity divided by the concentration). Total calcium and magnesium, as opposed to ionic calcium and magnesium, correlate poorly with neuromuscular irritability. Thus, in tears, measurement of these two divalent cations electrometrically is advantageous because there is no protein binding, no liquid junction effect, no sedimentation effect, and more confidence in activity coefficients, and tears reflect more closely the fluid environment of the brain through supply by the carotid. Typical concentrations of Ca.sup.2+ in tears are 0.4-0.8 mM (as compared to 1.09-1.33mM in plasma) and of Mg.sup.2+ in tears are 0.5-1.1 mM (as compared to 4.36-5.32 mg % in plasma). See Uotila et al., Invest. Ophthalmol. 11:258, 1972; Avisar et al., Invest. Ophthalmol. 16:1150, 1977.
It is therefore an object of the invention to provide a non-invasive method and device for obtaining tears, and for analyzing the tears for constituents.
It is another object of this invention to provide a method and device which maintains a constant flow of tears, driven by the constancy of the capillarity forces of a wick.
It is a further object of this invention to provide a method and device having embodiments which may be graded either semiquantitatively or quantitatively.
It is a further object of the invention to provide a non-invasive method and device which may use in the home or other non-clinical settings for the determination of organic and ionic constituents in tears.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.